Miniaturized 62Zn/62Cu generator for high concentration clinical delivery of 62Cu kit formulation for the facile preparation of radiolabeled Cu-bis(thiosemicarbazone) compounds

ABSTRACT

A new system accomplishes easy, interchangeable production of multiple PET radiopharmaceuticals through the use of a simplified eluant-only generator and a kit based synthesis technique employing lyophilized or freeze dried ligand. Thus, by simply switching the lyophilized ligand vial kit, any number of  62 Cu-labeled radiopharmaceuticals ( 62 Cu-ligand) can be interchangeably synthesized with only one  62 Zn/ 62 Cu generator.

This is a divisional application of U.S. patent application Ser. No. 10/571,202 filed Mar. 7, 2006, which is a 371 of PCT/US04/29252 filed Sep. 8, 2004, which claims the benefit of U.S. Provisional Application No. 60/501,156 filed Sep. 8, 2003.

Positron emission tomography (PET) is a highly sensitive imaging technique with many practical advantages over other radionuclide imaging modalities. Traditionally, its widespread clinical application has been limited by the economic burden associated with the purchase, operation, and maintenance of an in-house biomedical cyclotron required to produce the most commonly used short-lived PET radionuclides (¹⁵O, ¹³N, ¹¹C, and ¹⁸F). Even the well documented advantages of PET are not enough to offset these high expenses. Furthermore, the FDA regulatory issues regarding expedited production of short-lived radioisotopes using in-house cyclotrons are overwhelming.

A significant shift has occurred over the past decade in PET radiopharmaceutical production and distribution. Recognition of the potential of ¹⁸F, which can be produced using small cyclotrons and has a 110-minute half-life long enough for limited distribution, has led to the rise of several commercial PET radiopharmaceutical distribution chains. These efforts have focused on the glucose analog, ¹⁸F-FDG, distributed through regionally located cyclotron equipped pharmacies. Since each of these pharmacies can supply many local clinical facilities, the number of North American PET centers has grown substantially from 230 in 2000 to 603 in 2003. Also the vast majority of facilities now do not have a local cyclotron and rely exclusively on commercially manufactured and distributed ¹⁸F-FDG. Thus, PET has become a one tracer modality and methods of effective distribution of other tracers are lacking.

Substantial laboratory and clinical research suggests that various copper(II) bis(thiosemicarbazone) complexes can be useful as PET agents. A promising example, Copper(II) ⁶²Cu pyruvaldehyde bis(N4-methylthiosemicarbazone) or ⁶²Cu-PTSM, has been developed and has demonstrated utility as a myocardial, cerebral, renal, and tumor perfusion agent. This agent has a favorable short half-life of 9.7 minutes that reduces patient radiation dose and allows multiple serial studies during a single brief patient imaging session.

Furthermore, because ⁶²Cu is produced by a generator based on ⁶²Zn, with a half life of 9.3 hours, ⁶²Cu-PTSM can be readily distributed to hospitals through either regional or national distribution systems. Unlike local cyclotron production, regulatory organizations readily embrace distribution through use of a generator.

Another agent in the bis(thiosemicarbazone) family, ⁶²Cu ethylglyoxal bis(thiosemicarbazone) or ⁶²Cu-ETS is under investigation in human studies. Similar to ⁶²Cu-PTSM in structure, ⁶²Cu-ETS has shown more linear uptake at high blood flow rates and thus may provide a superior PET perfusion tracer for applications such as myocardial perfusion and renal blood flow measurements. Over the past several years, research with another bis(thiosemicarbazone) ligand, diacetyl-bis(N4-methylthiosemicarbazone) or H₂ATSM, has revealed that this compound, labeled with copper, has high promise as a hypoxia imaging agent. It has shown heterogeneous uptake in tumors with homogeneous perfusion images, strongly suggesting uptake reflecting hypoxic heterogeneity. Radiolabeled H₂ATSM also has produced “hot spot” myocardial images, reflecting hypoxia produced by experimental coronary occlusion. In addition several clinical studies have been reported in which Cu-ATSM tumor hypoxia images have correlated with prognosis and effectiveness of radiotherapy. Thus, ⁶²Cu-ATSM has the potential to be a very valuable tool by producing PET images which can guide treatment of tumors as well as provide assessment of cardiac and neurological disease. The short 9.7 minute half-life of ⁶²Cu makes it possible to combine multiple radiopharmaceuticals into one brief clinical imaging procedure. For example use of combined ⁶²Cu-PTSM imaging of tumor perfusion and tumor hypoxia in closely spaced studies using ⁶²Cu-ATSM could provide a far more quantitative and accurate evaluation of tumor hypoxia. Finally, ⁶²Cu radiopharmaceuticals can be distributed much more economically than non-generator produced ⁶⁰Cu, ⁶¹Cu, and ⁶⁴Cu.

As of now, by far, the largest application of nuclear imaging remains myocardial perfusion imaging in the diagnosis of coronary disease. Such imaging procedures account for more than 50% of all nuclear studies and are performed using single photon imaging which affords much poorer image resolution, less effective attenuation correction, and tracers based on ^(99m)Tc, which are less capable of tracking blood flow changes in the myocardium during stress. In order to realize the full potential of PET in this field, there is a clear need for distribution methods of tracers other than ¹⁸F-FDG, particularly effective perfusion tracers.

A modular ⁶²Zn/⁶²Cu generator has been developed which produces ⁶²Cu labeled agents in the bis(thiosemicarbazone) family via a method of in-line synthesis as described in U.S. Pat. No. 5,573,747. The 9.7 minute half-life of ⁶²Cu is long enough to facilitate radiopharmaceutical synthesis procedures and at the same time, it is short enough that multiple back-to-back imaging procedures are practical during a reasonably brief interval without interference of ⁶²Cu background activity from a previous injection. Also, such studies can be followed by another agent such as ¹⁸F-FDG after a reasonable delay, on the order of 40 minutes. The ability to perform back to back procedures is extremely beneficial because this is the preferred method of evaluation of myocardial blood flow. Such studies require regional comparison of myocardial uptake at rest with that during pharmacologic or exercise stress. The short half life of ⁶²Cu offers advantages for such procedures which are currently performed with the 6 hour half-life, ^(99m)Tc tracer.

The generator produced ⁶²Cu can be readily distributed to clinical facilities utilizing one of two distribution models. The 9.3 hour half-life ⁶²Zn parent (which decays to a daughter ⁶²Cu isotope) can be produced either in or near the ¹⁸F radiopharmacies using a 19 MeV cyclotron. Such a ⁶²Zn/⁶²Cu generator can then be delivered using the same local delivery network already in place for ¹⁸F. Alternatively, the ⁶²Zn parent can be produced and loaded into generators at a few large centralized facilities using >25 MeV cyclotrons and shipped to the local radiopharmacies or directly to hospitals.

There are many regulatory advantages to using a ⁶²Zn/⁶²Cu generator. Currently, almost all FDA approved radiopharmaceuticals are produced in a central commercial facility under well controlled conditions, and then distributed to local clinics where they are administered. Distribution via a generator system is a well accepted practice and the primary means of distribution of ^(99m)Tc, which is responsible for the majority of current nuclear medicine practice. Production of radiopharmaceuticals by numerous in-hospital cyclotron facilities is a concept which is not, and may never be, embraced by the FDA in any practical framework. In contrast, radionuclide generator systems like the ⁶²Zn/⁶²Cu generator of U.S. Pat. No. 5,573,747 are compatible with FDA accepted GMP production.

Although the inline synthesis generator as depicted in U.S. Pat. No. 5,573,747 has functioned very well in limited clinical studies, it has deficiencies which prevent it from being commercially viable on the large scale required for clinical use. Considering that any ⁶²Zn/⁶²Cu generator can be utilized for only one day, it is essential that every possible step be taken to simplify the system and thereby reduce the cost of production. Also, as to the generator of U.S. Pat. No. 5,573,747, the FDA has expressed a strong concern with regard to the generator septum which is entered repeatedly by the user. Instead, a product “collection directly into an empty sealed, pre-sterilized vial” is preferred and required for maintaining generator sterility. The generator's tubing set of that of U.S. Pat. No. 5,573,747 generator is costly to produce and the FDA also expressed reservations regarding the sterility during reuse. They stated “The product has a complex fluid path. To address this deficiency, you must add measurable tests to document the integrity of the system. However, given the design and recycling, it is doubtful that a test will be sufficient. A design modification may be needed.” In addition, inclusion of a pump inside the ⁶²Zn/⁶²Cu generator of U.S. Pat. No. 5,573,747 substantially increases the size of the generator housing and contributes to a higher shipping expense. Further, the transport of the large shield required for the 750 μl column is substantial in weight (35 lbs), and the shipping expense makes up a large portion of the cost for the generator system with a 1 day clinical life. Thus all means should be employed to reduce the weight and size of the ⁶²Zn/⁶²Cu generator. Another serious limitation is the large 33 mL injectable volume. Such a large volume is required because of the high salt content of the eluant solution, which must be diluted with sterile water for injection (SWFI) to achieve an isotonic solution. This large injection volume requires heavy, bulky shielding to avoid excessive technologist radioactive dose and precludes the convenient vial synthesis technique. Further, the high injection volume can also produce discomfort in some of the more sensitive patients and requires a prolonged injection time, which makes it difficult to define the input function required for typical PET quantitative analysis. Since in the ⁶²Zn/⁶²Cu generator of U.S. Pat. No. 5,573,747 the ligand addition is performed within the generator, only a single radiopharmaceutical can be produced without substantial added complexity. This limitation is unfortunate, particularly in light of the availability of the several very useful ⁶²Cu radiopharmaceuticals and the attractive applications of closely separated studies using two or more agents. For example, tumor or myocardial perfusion can be immediately followed with a ⁶²Cu-ATSM hypoxia scan. It can be readily speculated that such a perfusion scan is vital for achievement of a meaningful and quantitative hypoxia score.

There still exists a clear need for a system and method by which easy, interchangeable production of multiple PET radiopharmaceuticals can be accomplished.

A new system accomplishes easy, interchangeable production of multiple PET radiopharmaceuticals through the use of a simplified eluant-only generator and a kit based synthesis technique employing lyophilized or freeze dried ligand. Thus, by simply switching the lyophilized ligand vial kit, any number of ⁶²Cu-labeled radiopharmaceuticals (⁶²Cu-ligand) can be interchangeably synthesized with only one ⁶²Zn/⁶²Cu generator. In addition to interchangeable radiopharmaceutical production, use of a lyophilized kit formulation brings substantial benefits of higher stability and lower cost, as is well known in the industry. Further, the unit dose volume of the so produced radiopharmaceutical is greatly reduced, increasing patient comfort and administration time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a highly simplified and miniaturized generator that produces as an eluant, sterile, pyrogen-free ⁶²Cu²⁺ only.

FIG. 2 illustrates a loading apparatus which is used to load the generator of FIG. 1 with ⁶²Zn.

FIG. 3 illustrates the relation between flow rate of eluant from the generator of FIG. 1 and pressure introduced into the head space of the eluant vessel of the generator of FIG. 1.

FIG. 4 illustrates the reproducibility of flow rate of the generator of FIG. 1.

FIG. 5 illustrates the yield (⁶²Cu eluted/⁶²Zn on column) vs. bed volumes of eluant passed over the column of the generator of FIG. 1 (Z122).

FIG. 6( a) illustrates elution profiles of two generators, one loaded with 1.5 ml of 66.1 mCi ⁶²Zn solution (Z122) and the other loaded with 150 μl of 78.0 mCi ⁶²Zn solution (Z123), while FIG. 6( b) illustrates the elution profile of one of the generators (Z123) after 48 and 118 bed volumes of eluant passed over the column.

FIG. 7 illustrates the breakthrough levels of ⁶²Zn several generators at different loading of ⁶²Zn.

FIG. 8 depicts the chelation of a copper (II) ion, which is effective regardless of the substitution of side groups, R_(x) (where x=1, 2, 3, or 4).

FIG. 9 illustrates the thiol-mediated intracellular decomposition of CuII-PTSM that is believed to account for the prolonged “microsphere-like” tissue retention of the ⁶²Cu-radiolabel following intravenous administration of this and related ⁶²Cu-bis(thiosemicarbazone) complexes in normal cells.

FIG. 10 shows the reference absorbance spectra for a non-lyophilized solution of H₂PTSM (•) at a concentration of 0.67 μg/mL and for the Cu-PTSM (▪) solution formed by addition of 0.5 μg of CuCl₂ to the 3 mL cuvette.

FIG. 11 shows spectra from two representative lyophilized vials for the acetate excipient.

FIG. 12 shows spectra from two representative lyophilized vials for the dextrose excipient.

FIG. 13 shows spectra from two representative lyophilized vials for the trehalose excipient.

FIG. 14 shows TLC count profiles obtained for ⁶²CuPTSM on C18 and silica gel media and for the ionic ⁶²Cu²⁺.

A highly simplified and miniaturized generator is illustrated in FIG. 1 that produces as an eluant sterile, pyrogen-free ⁶²Cu²⁺ only. The reduced size of the generator column 10 facilitates the needed reduction in the volume of the eluted ⁶²Cu. The generator contains only a single eluant reservoir 1 and a small column 10 with a volume of 25-100 μL that is eluted by a modest pressurization in the sealed eluant reservoir 1. In a preferred embodiment, the column contains a beaded or particulate medium having a small volume of 25-100 μl. The pressurization streamlines and simplifies the loading and elution process and removes the peristaltic pump as the ⁶²Zn/⁶²Cu generator of U.S. Pat. No. 5,573,747 requires. The miniaturization of the column is possible because the mass of 50 mCi of ⁶²Zn is only 10 ng, which represents 0.8% of the binding capacity of just 1 μL of AG1X8 resin beads. This innovation accomplishes a 15 fold reduction in column volume, and together with use of modern tungsten shielding alloys 9 and the removal of the peristaltic pump, drastically reduces the weight of shielding needed around the column, leading to a sizable decrease in shipping expense. However, the most significant benefit of the column size reduction is that the injectable volume of the radiopharmaceutical product is reduced by a commensurate factor of 15. Since the salty 1.8 M NaCl, 0.2 M HCl eluant solution, together with a NaAc buffer, must be diluted six-fold to reach isotonicity, small elution volumes are essential for a lyophilized ligand kit synthesis approach. By effecting this large reduction in elution volume, the miniaturized generator or “microgenerator” permits production of an isotonic injectable PET radiopharmaceuticals in a receiving vial 17 of practical size (i.e. <5 mL).

The ⁶²Zn/⁶²Cu microgenerator is illustrated in FIG. 1 and contains four main parts: a resin microcolumn 10, an eluant vessel 1, a loading port 8 and an elution port 14. The key component of the system is the greatly size reduced borosilicate glass column. In one embodiment this column is 2.9 cm in total length, with an interior diameter of 2 mm and an outer diameter of 6 mm. The top and bottom of this column are formed into standard 8 mm vial closures. A 3 mm thick, 30 micron pore size glass frit is fused 5 mm from the bottom of the column. The column is filled to a calibration line with a 50 μl volume of AG1X8, 200-400 mesh anion exchange resin. Entry and exit from the column are provided by short pieces of 19.5 gauge (1 mm OD, 0.7 mm ID) corrosion resistant metal tubing (i.e., Inconel 625). To minimize void volume in the column, the tubing at the entry and exit of the column protrude through the 1.6 mm×7 mm Viton septum. A 0.5 mm hole in the septum facilitates the entry of the tubing which is introduced following crimp sealing with 8 mm aluminum crimp seals. The precise dimensions of the microcolumn 10 are not critical beyond the fact that the column is dimensioned to provide a single unit dosage of ⁶²Cu eluant which when mixed with SWFI yields an isotonic injectable solution of not greater than about 5 mL volume.

A “T” fitting above the column allows input connections from the eluant vessel 1 or the loading port 8 to the column 10 through the manipulation of pinch valves 5 and 6 operating on short sections of flexible tubing (i.e., Pharmed tubing [Cole-Parmer]). To assure sterility after autoclaving and to prevent contamination due to non-sterile fluids entering the system, a 0.22 μm hydrophilic (PVDF) sterilizing filter 4 a is placed in the load line. At the end of the load line, an HPLC fitting 7 allows simple, sterile connection and disconnection of the generator to the loading apparatus which is illustrated in FIG. 2. A pinch valve 11 of FIG. 1, controlled by a solenoid or manual actuator 12 is located below column 10. Opening and closing pinch valve 11 controls the elution process. Placement of the valve below the column 10 maintains constant pressure within the resin bed of the column and thereby prevents gas bubble formation produced by dissolved gases. Below this pinch valve is a rigid needle support 13 which assures the rigidity of needle 14 during its insertion through a septum of a kit vial 17. As FIG. 1 illustrates, a vial shield 15 is provided which has a removable top which has an entry port 16 positioned above the vial cavity within vial shield 15. The entry port provides access to the septum of vial 17 by the elution needle 14 of the generator as a lifting and alignment mechanism 18 moves a lyophilized ligand vial 17 within vial shield 15 into registration with elution needle 14.

The eluant vessel may be a 50 ml serum vial with a 20 mm grey butyl septum and aluminum crimp seal. The flow rate through the column is controlled by pressure maintained in the head space of this vessel. The bottle is pressurized by a pressure source 3 through a 0.22 μm hydrophobic (PTFE) sterilizing filter 4 b and sealed with a pinch valve 2 as shown in FIG. 1.

The loading apparatus is illustrated in FIG. 2 and is also set up for an air-pressure driven delivery system. A 3 ml conical Teflon vial 25 is used as the loading vessel. To modify this vessel for use as a loading vessel, a slightly undersized hole is drilled in the bottom and the vessel is heated to 100° C. After heating a 0.5 mm ID length of 1.6 mm OD tubing (i.e., Teflon) is quickly inserted into the opening. Upon cooling, the line is securely retained through thermal compression. This line passes over a shielded Geiger probe 28 (which is shielded 27) placed 2 cm below the loading vessel 25, which provided a rapidly responding indication of the presence or absence of radioactive solution in the tubing.

Two punctures are made in the loading vessel lid septum to allow the entrance of 0.5 mm ID tubing (i.e., Teflon). One is used to pressurize the loading vessel and the other is employed to transfer purified ⁶²Zn into the loading vessel. A pinch clamp 23 located on the air pressure line leading to the loading vessel 25 allows a pressure difference between the washing vessel 26 and the loading vessel 25 for filling of the interconnecting liquid flow path. Pressure applied from a pressure source 21 is accurately measured using a digital pressure transducer 22 (i.e., Omega PX170). Connections between the pressure source to the washing and loading vessels are made using 0.5 mm ID Teflon tubing. Tygon tubing (1.5 mm ID) connects the pressure transducer to the loading pressure system.

The washing vessel 26, like the loading vessel 25, has two punctures in the septum for the insertion of 0.5 mm ID tubing. One line pressurizes the washing vessel and the other line supplies 2M HCl to the loading line. The lines that flow from the loading vessel and the washing vessel terminate in a 3-way HPLC selector valve 29. This valve allows flow from the washing vessel to the loading vessel, for liquid line filling, from the loading vessel to the loading port 8 of the generator (FIG. 1), for generator loading, and from the washing vessel to the loading port 8 of the generator for liquid line filling and generator loading. At the end of the load line on the loading apparatus, an HPLC quick connect 30 is provided for connection to the generator load port 8.

A load activity (i.e., ⁶²Zn), is pumped into the conical loading vessel 25 of FIG. 2, following preparation of the loading system, which includes filling of all lines with 2M HCl from the washing vessel. The loading procedure is as follows. The pinch clamp 23 is closed under atmospheric pressure to facilitate filling of the line originating at the 3-way HPLC valve 29 and ending at the loading vessel 25 with 2M HCl from the washing vessel 26. The 3-way valve is then turned to select the flow path from the washing vessel to the load line to liquid fill that line as well. The output 30 of the load apparatus is connected to the loading port 8 of the generator in a manner assuring liquid continuity with no air bubbles. The pinch clamp 5 leading from the eluant vessel 1 to the “T” fitting is closed to prevent flow of ⁶²Zn into the eluant vessel, and 2M HCl is then delivered from the washing vessel 26 (FIG. 2) through the generator column 10 to verify that the desired loading flow rate is achieved. The 3-way valve is switched to direct ⁶²Zn solution from the loading vessel 25 to column 10 of the generator. During the loading procedure, an eventual rapid drop in Geiger probe 28 reading signals a switch of the 3 way valve from the loading vessel to the washing vessel. The column 10 of the generator is then washed with 256 μl of eluant, after which the loading vessel is detached and the column 10 of the generator is ready for use.

All of the bis(thiosemicarbazone) derivatives readily and avidly chelate Cu in the same manner. FIG. 8 depicts the chelation of a copper (II) ion, which is effective regardless of the substitution of side groups, R_(x) (where x=1, 2, 3, or 4). This is because the Cu⁺² ion interacts with the hybrid orbitals of nitrogen and sulfur atoms in the compound in a manner little influenced by the composition of R_(x). The conjugated π-system within the ligand is conserved in this reaction and the substituted groups, which are outside of this conjugated system, do not participate in the mechanism of chelation. When the copper ion binds to the sulfur atoms, there is a rearrangement of electrons, which results in the formation of two nitrogen-carbon double bonds and the loss of two H+ atoms. In the complexed ligand, the copper atom is bound in a square configuration which is common for copper in its 2+ state. Table 1 is a partial list of bis(thiosemicarbazone) complexes which have been reported in the literature.

The structure of the bis(thiosemicarbazone) complexes is as follows:

TABLE 1 Partial list of Copper bis(thiosemicarbazone) complexes R₁ R₂ R₃ R₄ GTS H H H H GTSM H H CH₃ H PTS CH₃ H H H PTSM CH₃ H CH₃ H PTSM₂ CH₃ H CH₃ CH₃ ETS CH₂CH₃ H H H ETSM CH₂CH₃ H CH₃ H ETSM₂ CH₂CH₃ H CH₃ CH₃ PTSE CH₃ H CH₂CH₃ H PTSP CH₃ H C₆H₅ H ATS CH₃ CH₃ H H ATSM CH₃ CH₃ CH₃ H CTS CH₂CH₃ CH₃ H H CTSM CH₂CH₃ CH₃ CH₃ H DTS CH₂CH₃ CH₂CH₃ H H DTSM CH₂CH₃ CH₂CH₃ CH₃ H

TABLE 2 Partition coefficients of selected Cu bis(thiosemicarbazone) complexes R₁ R₂ R₃ R₄ log P* ATSM CH₃ CH₃ CH₃ H 2.26 PTSM CH₃ H CH₃ H 1.92 ETS CH₂CH₃ H H H 1.35 From measured octanol/water partition coefficients, P, of the corresponding ⁶⁷Cu-complexes.

Although the chelation chemistry of these compounds is very similar, they exhibit very different chemical and physical properties in vivo due to their differing side Rx groups. For example, H₂ATSM and H₂PTSM differ by only the R₂ sidegroup but have octanol/water partition coefficients that differ by 0.34 (Table 2). Different side group substitutions can also substantially alter the binding of the copper compound to various components of the blood. Such effects have been shown to be highly species dependent. In particular, Cu-PTSM exhibits low binding to blood in most animal species other than humans, but exhibits significant binding in humans, which causes non-linear uptake at high flow levels. The variant Cu-ETS, on the other hand, has low reversible binding to blood plasma and thus proves to be a more effective perfusion tracer than Cu-PTSM in applications in which high blood flow organs such as heart and kidney are imaged.

FIG. 9 illustrates the thiol-mediated intracellular decomposition of CuII-PTSM that is believed to account for the prolonged “microsphere-like” tissue retention of the ⁶²Cu-radiolabel following intravenous administration of this and related ⁶²Cu-bis(thiosemicarbazone) complexes in normal cells. The uncharged lipophilic copper(II) bis(thiosemicarbazone) complexes readily diffuse across cell membranes, whereupon they are susceptible to reductive decomposition by reaction with ubiquitous intracellular thiols, such as glutathione. Electron transfer from the thiol sulfur to the CuII-bis(thiosemicarbazone) complex leads to dissociation of CuI from the bis(thiosemicarbazone) ligand to become bound (and effectively trapped) by intracellular macromolecules. Different CuII-bis(thiosemicarbazone) complexes have distinct reduction potentials, which significantly effects intracellular trapping. CuATSM, for example, has a lower redox potential than CuPTSM or CuETS at 100 mV. As a result it is not reduced in normoxic tissues and therefore quickly washes out. However, in hypoxic tissues, the Cu(II) of CuATSM is quickly and irreversibly reduced to Cu(I) and retained due to the lack of electron carrier (Oxygen).

The new method for synthesizing radiolabeled Cu-bis(thiosemicarbazone) compounds centers on the utilization of a lyophilized form of the ligand. Lyophilization or freeze drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to sublime directly from solid to vapor without passing through a liquid phase. This process can effectively prevent the crystallization of the solute thereby rendering it far more readily soluble than normally would be the case in solid form. After sterile lyophilization, the dry resulting cake remains sterile and can be stored for a much longer period of time than if it had remained in an aqueous solution as is very well known in the pharmaceutical industry. Solutions containing the ligand of interest are lyophilized with an excipient and stored in 2-5 ml vials. The vial contents can be virtually instantaneously rendered soluble (reconstituted) by simple addition of sterile water for injection (SWFI) or with SWFI containing a buffer agent such as Sodium Acetate. Sterile radioactive Cu⁺² solution from the ⁶²Zn/⁶²Cu generator or other source can then be added to the vial for instantaneous formation of the radiolabeled Cu-ligand complex. Such instant synthesis is essential for practical clinical use of such a short-lived agent as ⁶²Cu (t_(1/2)=9.7 min). In the case that ⁶²Cu is obtained from the ⁶²Zn/⁶²Cu generator the solution optimally consists of 1.8M NaCl, 0.2M HCl and the vial contents most optimally contain a 2 molar excess of sodium acetate or other buffer and a volume of SWFI which together with the ⁶²Cu bearing solution brings the osmotic pressure of the final injectable solution to a value near that of blood (7.7 atm) and produces a pH most optimally in the range 5.5-7.0.

Studies evaluating the compatibility of the bis(thiosemicarbazone) ligands with lyophilization based kit synthesis were conducted using H₂PTSM as a model compound. The first of these studies evaluated the feasibility of ligand lyophilization and rapid reconstitution. The entire class of bis(thiosemicarbazone) compounds is very insoluble in water. Consequently, in order to prepare a principally aqueous solution for lyophilization, the crystalline form of the H₂PTSM was dissolved at the maximum concentration in dimethylsulfoxide (DMSO), (200 mg/mL H₂PTSM). Because of high ligand solubility in DMSO, very small volumes of this solution added to water produced a nearly DMSO-free aqueous solution of the ligand having a concentration of 2 μg/mL. To minimize the possibility of ligand precipitation, the DMSO solution was added to hot water (90° C.) during rapid stifling. This solution was then cooled to room temperature and assayed for potency using a spectrophotometer. The very small amount of DMSO in the aqueous solution is expected to sublimate along with water during lyophilization. In order to ensure maximum potency after lyophilization, an excipient (either dextrose [6.7 mg/mL], trehalose [6.7 mg/mL] or sodium acetate [5.4 mg/mL]) was added. The purpose of the excipient was to prevent the ligand from crystallizing out by maintaining dispersion in the cake formed during lyophilization. The sodium acetate performed the added function of buffering the final product to acceptable pH upon addition of 0.1-0.15 mL of ⁶²Zn/⁶²Cu generator eluted solution. Any true crystallization of the ligand must be avoided because such crystallized forms are extremely difficult to bring back into aqueous solution. The solution consisting of 2 μg/mL ligand together with excipient was aliquotted 1 ml per 2-mL vial and sent for lyophilization. The lyophilization of the vials was performed by first freezing the samples to −70° C. for a 2-hour period then equilibrating the vials (in the lyophilizer) at −20° C. for two hours. Finally, refrigeration was terminated and an 18-hour vacuum (25-50 mtorr) cycle was started. At the end of the vacuum cycle the vials were crimp-sealed under nitrogen gas.

EXAMPLES Example 1

To evaluate the pressure method of delivery of eluant through the generator column, the pressure in the elution vessel was varied and the flow rate was determined by measuring the eluted volume per unit time. The desired elution flow rate is estimated by scaling from the 750 μL column generator, which elutes at 3.6 ml/min. By scaling this flow rate in accordance with the ratio of resin bed volumes in each design, a 240 μl/min flow rate for the new system was predicted. The eluant vessel initially contains 5 ml of eluant. With a maximum estimated clinical column usage of 20 elutions with approximately 0.12 ml per elution volume, there is a 2.4 ml reduction in eluant volume, producing only a 5% change in elution pressure and therefore a negligible variation of flow rate over a day of clinical use, which is the maximum shelf life in routine clinical use.

The method of pressure driven elution achieved the targeted elution rate of 240 μl/min. The eluant vessel was empirically pressurized over a range of pressures between 5-15 psig. The eluted samples were then weighed to determine the flow rate under a specific pressure. In order to investigate the effect of variable flow rate on column performance, elution profiles at various pressures were collected in fractions of 5 or 10 seconds over 30, 60, or 120 second periods. All elutions and fractions were immediately measured in a Capintec CRC-15R and decay corrected to the beginning of elution. These elutions and fractions were also weighed to determine the volume eluted per unit time.

Preliminary pilot studies of the simplified microgenerator and vial synthesis approach were performed. A primary purpose of these studies was to demonstrate the production feasibility and acceptable performance of a generator column of greatly reduced physical size and volume. Secondarily, the feasibility of simple methods of column elution with passive pressure, rather than a pump, were explored.

A total of four columns were loaded and evaluated. All four columns were loaded at a flow rate of 10 μl/min. Variable load volumes were assessed to evaluate the effect on breakthrough and yield performance. Volumes ranged from 0.15 ml to 1.5 ml. Load activity was sequentially increased to assess any potential radiation damage effects on the performance of the column. Activity ranged from 2.9 mCi to 78 mCi.

Breakthrough of selected eluted samples was counted following full decay of ⁶²Cu in a NaI well counter (Searle model 1197) (4 hours post elution). Purified ⁶²Zn was used to calibrate the system. Breakthrough is reported as the ratio of ⁶²Zn in the eluant solution divided by the ⁶²Zn on the generator column, both decayed to the same point in time.

Four prototype microgenerators were constructed. Each employed a 50-mL glass eluant vessel directly connected by tubing to a 50-μL resin-filled column, which was coupled to an output tubing line. Elution was controlled by opening and closing a calibrated manual pinch clamp. The clamp was designed and adjusted to avoid exerting excess pressure on the tubing. To pressurize the eluant reservoir, a pressure source was connected to the tubing line leading to the head space of the vessel (see FIG. 1; 3). In order to demonstrate the feasibility of pressure-driven flow and to establish the relation between pressure and flow rate, elution samples were collected and weighed for vessel pressures ranging from 0-15 psig. In addition, careful studies were performed to establish the flow rate reproducibility. A solution of ⁶²Zn was loaded onto each column at a load rate of 10 μL/min. To assess the potential radiation effects on the very small resin bed, activities ranging from 2.9 mCi to 78 mCi were explored. The effect of load volume, which ranged from 0.15 mL to 1.5 mL, on ⁶²Cu yield and ⁶²Zn breakthrough was also investigated. For each generator, elution samples of 30 and 60 second duration were collected frequently over a period of two days. Serial elution samples were also collected in 5- or 10-second fractions. Sample activity levels were assayed in a dose calibrator (Capintec CRC-15) and decay corrected to obtain elution profiles and yield estimates. Following decay of ⁶²Cu (>4 hrs post elution), samples were counted in a NaI to measure breakthrough.

In flow rate studies, results indicated that pressures of 1-2 psig were adequate to achieve the desired flow rates of 50-200 μL/30 sec. At such low pressures, however, changes in temperature and eluant volume produced significant changes in flow rate. In order to attenuate the impact of these factors, vessel pressure was increased roughly ten-fold, and studies of the pressure-flow rate relation were repeated for pressures of 5-15 psig. Corresponding increases in the length of the small I.D. tubing between the eluant vessel and the column were incorporated to maintain the desired flow rates at these higher pressures. As shown in FIG. 3, flow rate and vessel pressure displayed a very tight linear relation. Thus, the optimal flow rate, once established, clearly can be achieved through choice of the corresponding vessel pressure. Studies also demonstrated that pressure-driven flow is highly reproducible, as illustrated by the narrow peak in FIG. 4. At 10 psig, a 30-second elution dispensed a mean volume of 106.2 μL with a standard deviation of <1%.

The equation shown in FIG. 3 and the ideal gas law may be used to estimate the effect of changes in eluant volume and temperature on the volume delivered in a 30 second elution. Assuming an initial vessel pressure of 10 psig, which produces a flow rate of ˜106 μL/30 sec, and an initial temperature of 77° F., a decrease in temperature of 10° F. would lower delivered volume by only 4.0 μL. A decrease in eluant volume of 2.12 mL, which corresponds to twenty 106 μL elutions, would lower delivered volume of roughly 9.7 μL. Such changes in eluant volume and ambient temperature represent fairly extreme conditions of clinical use. Even in the unlikely event that they occur together, they would produce only modest changes (<13%) in delivered volume, which is reasonable in the context of clinical applications.

Prototype microgenerators performed very well in pilot studies. Table 3 lists the yield and breakthrough data for the four microgenerators and a typical current clinical generator. For all of the prototype generators, the average yield values (⁶²Cu eluted/⁶²Zn on column) for the first 30 seconds of elution were equivalent to or greater than current generator yields of roughly 50%. These results clearly support the hypothesis that adequate yield can be obtained using a miniaturized column. Furthermore, as shown in FIG. 5, high yields were maintained under conditions of frequent elution throughout the two day testing period.

Pilot studies revealed multiple factors that affect generator performance. Firstly, over time, as more eluant was washed over the resin column, yield increased (see FIG. 5). Secondly, reduced load volume may initially produce lower yield. For example, generator Z123 was loaded with a ten-fold smaller volume than Z122 (150 μl vs. 1.5 ml) and also produced a roughly 10% lower average yield (see Table 1). Presumably, under conditions of lower load volume, the ⁶²Zn activity binds to the resin in a narrow band close to the top of the column. As a result, a greater volume must flow over the resin before the ⁶²Cu reaches the column output. Elution profiles (see FIG. 6) support this hypothesis. In these profiles, activity concentration is plotted against eluted volume. As shown in FIG. 6( a), Z123 shows a much later activity peak than Z122. As a result, for generators with low load volume, more of the peak and tail are cut off, in a 30-second elution, resulting in a lower yield. As suggested by FIG. 5, this yield may rise after adequate eluant has passed over the resin bed. FIG. 6( b) displays elution profiles for Z123 taken after 48 and 118 bed volumes of eluant had been passed over the column. As shown, the later profile shows an earlier and narrower peak.

TABLE 3 Summary performance data for pilot microgenerators Load ⁶²Zn breakthrough Activity Load Volume Average 30 s fraction Gen. ID (mCi) (ml) % yield (Initial levels) Typical 150 ~9 49-55% 2.60 × 10⁻⁷ current generator Z120 2.9 0.95 63% N/A Z121 18.4 1.0 76% 3.54 × 10⁻⁷ Z122 66.1 1.5 65% 2.56 × 10⁻⁷ Z123 78.0 0.15 54% 3.77 × 10⁻⁸

The results in Table 3 also show that breakthrough of the ⁶²Zn parent isotope can be maintained well below levels achieved with the modular generator of U.S. Pat. No. 5,573,747. Microgenerators that were manufactured with larger load solution volumes (i.e. Z121-122) showed breakthrough that was comparable to that of current clinical generators. However, generator Z123, which was loaded using a smaller volume of more concentrated ⁶²Zn solution, produced an order of magnitude reduction in initial breakthrough levels. Lower load volume also proved advantageous for maintaining low breakthrough levels (see FIG. 7). Z123, which was loaded with the lowest volume, showed, at most, a 2-fold increase in breakthrough after passage of ˜40 bed volumes of eluant (˜20 elutions) compared to an increase of approximately 10-fold for Z121 and Z122.

In summary, the results of pilot studies showed that a dramatically miniaturized column can perform as well as current generators with respect to important parameters, such as yield and breakthrough, and that such performance can be maintained over the course of expected clinical use. They also suggest a scaled down column may even enable superior performance. Pilot study results also showed that pressure-driven elution was highly reproducible and that variability in delivered volume would be within acceptable limits. Taken together, these findings strongly support the feasibility of the proposed microgenerator design.

Example 2

Tests were run on randomly selected lyophilized vials to compare the concentration of the ligands before lyophilization and after reconstitution. A reliable and sensitive technique for measurement of bis(thiosemicarbazone) ligand concentration was made using UV/VIS spectroscopy. This technique is based upon the ligand's avid chelation of ionic copper and the distinct visible absorbance peak of the resulting copper compound. The contents of each vial were reconstituted with 1.0 mL of deionized water and were diluted to 3.0 mL in a quartz spectrophotometer cuvette, resulting in a concentration of 0.67 μg/mL based on 100% recovery into solution. An excess of CuCl₂ (1 μg) was added to permit quantitative formation of Cu-PTSM. This process was performed quickly (2-3 min), and the UV/VIS spectra was measured within 30 seconds. In this manner, the feasibility of rapid reconstitution was assessed. Spectra were obtained for the Cu-PTSM in the three excipient solutions (dextrose, trehalose, or sodium acetate) and for a Cu-PTSM reference solution without excipient.

A second study evaluated the feasibility of in-vitro radiolabeling of reconstituted lyophilized ligand with microgenerator-produced ⁶²Cu²⁺ to produce ⁶²CuPTSM with high radiochemical purity. Formation and purity of Cu-PTSM was assessed using thin layer chromatography (TLC). Lyophilized H₂PTSM (2 μg) and trehalose excipient were reconstituted in the lyophilization vial with 105 μL of 0.4 M sodium acetate (i.e. buffer) and 1.5 mL of DI water, thus providing a 2-fold molar excess of buffer and water dilution necessary to reach isotonicity upon addition of 105 μl of generator eluant (1.8 M NaCl, 0.2 M HCl). The vial was vortexed for 30 seconds and then was left to sit undisturbed for 10 minutes. The vial's rubber stopper was removed, and the microcolumn was eluted to deliver 105 μL directly into the vial. The solution mixture was then gently swirled for a few seconds and left to sit at room temperature for 30 seconds. Immediately thereafter, duplicate 0.5 μL aliquots were spotted at the 1.0 cm mark on C18 and silica gel TLC plates. The glass plates were immediately placed in the development tank and were developed for 30 minutes in 100% ethanol mobile phase. In addition, reference ionic ⁶²Cu²⁺ plates were run separately. In this test, any ionic ⁶²Cu remains at the origin whereas the lipophilic ⁶²CuPTSM compound travels with the solvent. A straw detector-based scanner was used to count the activity distribution on each track of each plate. A minimum of 10,000 counts was acquired for each track.

Using the techniques described above, lyophilized vials, containing cakes of ligand and excipient, were produced. Production was successfully accomplished using all three test excipients. Furthermore, feasibility studies of rapid reconstitution yielded exceptional results. Absorbance spectra are shown in FIG. 10-FIG. 13. FIG. 10 shows the reference absorbance spectra for a non-lyophilized solution of H₂PTSM (•) at a concentration of 0.67 μg/mL and for the Cu-PTSM (▪) solution formed by addition of 0.5 μg of CuCl₂ to the 3 mL cuvette. As may be seen in the figure, following addition of ionic copper, the H₂PTSM absorbance peak (320 nm) disappears and is replaced by the characteristic absorbance peaks of Cu-PTSM (462 nm and 308 nm), demonstrating complete conversion of H₂PTSM to Cu-PTSM. The effectiveness with which H₂PTSM was reconstituted from the lyophilized vials may be assessed through comparison with this reference Cu-PTSM spectra. FIG. 11, FIG. 12, and FIG. 13 shows spectra from two representative lyophilized vials for the acetate, dextrose, and trehalose excipients, respectively. For comparison, each panel also includes the non-lyophilized reference Cu-PTSM spectrum from FIG. 10. As shown, for all three excipients, the spectra obtained from lyophilized ligand are virtually identical to the reference spectrum. These results show that lyophilized ligand can be completely and rapidly reconstituted.

FIG. 14 shows TLC count profiles obtained for ⁶²CuPTSM on C18 and silica gel media and for the ionic ⁶²Cu²⁺. As expected, for ⁶²CuPTSM profiles [see FIG. 14, (a) and (b)], a single mobile radioactive species was observed, as demonstrated by the presence of a single peak on each track. Rf values, averaged across the two tracks, were 0.49 and 0.72 for C18 and silica gel, respectively. These values agree with Rf values that have consistently been obtained for ⁶²CuPTSM produced using other methods. In contrast, as shown in FIG. 14( c), ionic copper remained at the origin on both media, and Rf's for ionic ⁶²Cu²⁺ were 0.23 and 0.19 for C18 and silica gel, respectively. The absence of an origin peak on the ⁶²CuPTSM profiles [FIG. 14, (a) and (b)] shows that essentially no ionic copper remained following combination with the ligand. Average radiochemical purity of ⁶²CuPTSM, based on the TLC profiles (n=4), was 96%.

The ⁶²Zn/⁶²Cu microgenerator and lyophilized ligand kit can play a significant role in advancing clinical PET imaging by serving as a distribution source of a short-lived PET isotope for synthesis of a wide variety of radiopharmaceuticals. The microgenerator together with kit synthesis techniques fit seamlessly into the current regulatory and commercial paradigm of distributable radiopharmaceuticals. One center can process the bombarded target, perform necessary radiochemistry to purify the ⁶²Zn, and load large numbers of generators. Since interchangeable kits can be employed with the same generator, the synthesis technique is both flexible and economical. The ⁶²Zn/⁶²Cu microgenerator and lyophilized ligand kit can play a major role in advancing clinical PET imaging in oncology, cardiology, and neurology. Finally, the miniaturization of the generator facilitates delivery and labeling in clinically convenient dose volumes. 

I claim:
 1. A method of producing radiopharmaceutical agents for use in positron emission tomography comprising: providing a column having a small volume capacity; filling a portion of said column with anion exchange resin beads having a small volume between 25 and 100 μl; introducing a ⁶²Zn solution into the column to bond the radioactive isotope to the beads; providing a septum closed vial containing lypholized ligand compound; soluablizing the lypholized compound in the vial by introducing a reconstitution agent; providing a pressurized eluant vessel in fluid communication with the column; releasing eluant from the eluant vessel to flow into the column and through the resin beads to a dispensing point; directing the eluant from the column dispensing point into the vial thereby forming a radiolabeled Cu-ligand compound.
 2. The method of claim 1 wherein the compound comprises at least one bis(thiosemicarbazone) compound represented by the following formula:

wherein R₁ and R₂ are independently hydrogen or a hydrocarbyl group; R₃ is hydrogen, a hydrocarbyl or an aryl group; and R₄ is hydrogen or a hydrocarbyl group, wherein any hydrocarbyl or aryl group optionally contains a heteroatom from the group of O, N, S, P, and Si in place of a carbon atom or a halogen atom in place of a hydrogen atom, together with a suitable excipient.
 3. The method of claim 1 wherein the reconstitution agent is sterile water.
 4. The method of claim 1 wherein the reconstitution agent comprises a buffering agent.
 5. The method of claim 4 wherein the buffering agent is sodium acetate.
 6. The method of claim 1 wherein the step of providing a lyophilized form of the ligand compound comprises freezing the compound; placing the compound under a vacuum; and removing the water from the compound.
 7. The method of claim 1 wherein the radiolabeled Cu-ligand compound comprises a ⁶²Cu-ligand.
 8. The method of claim 7 wherein the reconstitution agent comprises 1.8M NaCl and 0.2M HCI.
 9. The method of claim 7 wherein the reconstitution agent comprises a 2 molar excess of a buffer.
 10. The method of claim 9 wherein the buffer is sodium acetate.
 11. The method of claim 1 wherein the dispensing outlet point is a needle tip.
 12. The method of claim 1 wherein for each production of a radiopharmaceutical 0.1 to 0.3 mL of eluant is released through the column at a flow rate of 100-500 μL/minute.
 13. A method for producing a radiopharmaceutical comprising the steps of: releasing 0.1 to 0.3 mL of an eluant from an elution vessel to flow through a column containing resin beads having a ⁶²Zn radioactive isotope to a needle tip; wherein the eluant is released through the column at a flow rate of 100-500 μL/minute; directing said eluant from the outlet point into a septum closed vial containing a water reconstituted lyophilized bis(thiosemicarbazone) compound of the formula

wherein R₁ and R₂ are independently hydrogen or a hydrocarbyl group; R₃ is hydrogen, a hydrocarbyl or an aryl group; and R₄ is hydrogen or a hydrocarbyl group and wherein any hydrocarbyl or aryl group optionally contains a heteroatom from the group of O, N, S, P, and Si in place of a carbon atom or a halogen atom in place of a hydrogen atom, to form an injectable solution.
 14. A method for producing a radiopharmaceutical comprising the steps of: releasing an eluant from an elution vessel to flow through a column containing resin beads having a bound radioactive isotope to a dispensing outlet point; directing said eluant from the outlet point into a septum closed vial containing a water reconstituted lyophilized bis(thiosemicarbazone) compound of the formula

wherein R₁ and R₂ are independently hydrogen or a hydrocarbyl group; R₃ is hydrogen, a hydrocarbyl or an aryl group; and R₄ is hydrogen or a hydrocarbyl group and wherein any hydrocarbyl or aryl group optionally contains a heteroatom from the group of O, N, S, P, and Si in place of a carbon atom or a halogen atom in place of a hydrogen atom, to form an injectable solution.
 15. The method of claim 14 wherein the dispensing outlet point is a needle tip.
 16. The method of claim 14 wherein the bound radioactive isotope is ⁶²Zn.
 17. The method of claim 14 wherein the eluant carries ⁶²Cu to the dispensing outlet point.
 18. The method of claim 14 wherein for each production of a radiopharmaceutical 0.1 to 0.3 mL of eluant is released through the column at a flow rate of 100-500 μL/minute.
 19. The method of claim 13 wherein said resin beads have a small volume between 25 and 100 μl.
 20. The method of claim 14 wherein said resin beads have a small volume between 25 and 100 μl. 